In vitro method for identifying target sites for antisense-mediated inhibition of a selected gene

ABSTRACT

A method for making a directed antisense library against a target transcript is described. A cDNA of the target transcript is cloned in an appropriate cloning vector. Next, a plurality of deletion derivatives of the cloned cDNA is prepared such that the deletions serially extend into the cDNA from one end thereof. The resulting deletion library is then treated such that cDNA is removed from the other end of each cDNA insert, thus obtaining a fragment library having fragments of a selected size. An antisense gene is then inserted into each fragment of the fragment library, resulting in the directed antisense library. An illustrative antisense gene in the hammerhead ribozyme catalytic core. Plasmids for making the antisense library, plasmids and methods for making the fragment library, and a method for identifying target sites for antisense-mediated gene inhibition are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a division of U.S. Ser. No. 09/647,344, filedDec. 4, 2000, which is an application filed under 35 U.S.C. §371 ofPCT/US99/06742, filed Mar. 28, 1999, which claims the benefit of U.S.Provisional Application No. 60/079,792, filed Mar. 28, 1998, and U.S.Provisional Application No. 60/107,504, filed Nov. 6, 1998.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant No.1R03RR08849 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] This invention relates to antisense agents. More particularly,the invention relates to compositions and methods for generation ofdirected antisense libraries and methods of use thereof wherein theantisense agents in the libraries can potentially bind to every bindingsite on a selected RNA transcript.

[0004] Antisense RNA, DNA, and ribozymes have been widely studied asresearch tools and potential therapeutic agents for inhibiting theexpression of specific genes. These agents operate by binding to acomplementary region on an RNA transcript produced from the gene ofinterest. On binding, the antisense agent can prevent expression of theRNA, and this can occur through a variety of different mechanisms. Thereare many sites on any given RNA for targeted inhibition by an antisensemolecule. For a typical RNA transcript of 2000 nucleotides, just under2000 target sites are available. Examination of a few to tens ofrandomly chosen target sites reveals a great variability in activity.Clearly, not all target sites are equivalent in their ability to permitantisense mediated inhibition. Consequently, identification of effectivetarget sites on the RNA transcript for interaction with the antisensemolecule is imperative for successful application of antisensetechnology. Methods currently available for this purpose include the useof computer algorithms to predict target accessibility based on thepredicted secondary structure of the mRNA, the use of randomizedoligonucleotide and ribozyme libraries in cell free systems, and theexamination of a few to tens of antisense oligonucleotides, targeted toarbitrarily chosen sites, in cell culture assays. These approaches havemet with limited success.

[0005] To identify the most effective target site(s), the followingconditions should be met. First, all possible sites on the target RNAshould be evaluated. Second, evaluation should be carried out in thenormal cellular milieu. This insures that the target is in its naturalstructure, associated with its normal complement of cellular factors.Additionally, the antisense agent has the opportunity to act onalternate structures that may arise as a result of the many RNAprocessing reactions.

[0006] To evaluate all target sites, antisense libraries must be used.These libraries should contain antisense molecules targeted to everysite. One approach is the use of completely randomized DNA, RNA, orribozyme libraries. The use of completely randomized libraries suffersfrom two major disadvantages. First, while such libraries may containantisense molecules directed at all sites on the target RNA, they alsocontain antisense molecules directed at all sites of all potential RNAtranscripts produced by the cell. Therefore, these random librariespotentially have the capability to inhibit expression of every gene inthe cell. Because of this, random libraries are limited to in vitro usein cell free assays. Second, the complexity of these libraries isenormous. For example, a random library that uses 14 nucleotides torecognize its target must contain at least 2.6×10⁸ (i.e., 4¹⁴) differentmembers. Realistically, the size of the library must be at least 10- to100-fold greater in size to insure representation of all sequences. Theproduction and screening of such large libraries is likely beyondcurrent capabilities.

[0007] Herein there is described a new method for identifying optimalantisense target sites against any desired RNA transcript. This is adirected library approach. In other words, this approach uses anantisense library that targets every site on any selected RNA and onlysites present on the selected RNA. This library, therefore, does notinhibit other non-target RNA transcripts. This approach is also animprovement over known methods because it uses relatively smalllibraries. For example, a library targeting an RNA transcript of 2000nucleotides, and using 14 nucleotides to recognize its target,theoretically needs 1986 members. In practice, the library would need tobe 10- to 50-times this size. At 50 times, or 99,300 members, this isstill a relatively small library. These directed libraries can be usedin both in vitro and in vivo assays for the detection of effectivetarget sites for antisense mediated gene inhibition.

[0008] In view of the foregoing, it will be appreciated that a methodfor generating directed antisense libraries would be a significantadvancement in the art. Herein is described a method for examining theentire length of any RNA transcript for sites that are accessible toantisense agents. This approach allows for the localization of the mosteffective sites for targeting with antisense agents.

BRIEF SUMMARY OF THE INVENTION

[0009] It is an advantage of the present invention to provide a simpleand inexpensive method for producing directed antisense librariesagainst any selected RNA transcript.

[0010] It is also an advantage of the invention to provide a method ofproducing directed antisense libraries wherein such libraries containantisense agents directed against all targets spanning the entireselected RNA transcript.

[0011] It is another advantage of the invention to provide a method ofusing directed antisense libraries for locating efficient target siteson the selected RNA transcript.

[0012] It is still another advantage of the invention to providecompositions for use in constructing directed antisense libraries.

[0013] It is yet another advantage to provide a method for makingfragment libraries of a selected size of DNA fragment inserted in acloning vector.

[0014] These and other advantages can be addressed by providing a methodfor generating an antisense library targeted to a selected RNAtranscript comprising:

[0015] (a) preparing a double-stranded cDNA, comprising a first end, asecond end, and a central site thereof, from the selected RNA transcriptand cloning the cDNA in a cloning vector comprising a promoterconfigured such that an antisense transcript of the cDNA is synthesizedupon transcription mediated by the promoter, resulting in a cloned cDNA;

[0016] (b) creating a plurality of deletion derivatives of the clonedcDNA wherein each of the plurality of deletion derivatives has adeletion extending from the first end into the cloned cDNA such that theplurality of deletion derivatives comprises a deletion librarycomprising deletions extend serially into the cDNA;

[0017] (c) reducing the size of the cDNA contained in the deletionlibrary to a preselected size by removing a portion of the cDNA from thesecond end thereof to result in a fragment library;

[0018] (d) inserting an antisense gene DNA into the central site of thecDNA in the fragment library, thereby obtaining the antisense library.

[0019] Illustrative cloning vectors comprise multi-cloning sequencescomprising SEQ ID NO:1 and a combination of SEQ ID NO:2 and SEQ ID NO:3.In an illustrative embodiment of the invention, the deletion derivativesare created with exonuclease III resection of the cloned cDNA.

[0020] The size of the cDNA contained in the deletion library isillustratively reduced to a preselected size by digesting the deletionlibrary with a type IIS restriction endonuclease. Further, inserting theantisense gene DNA into the central site of the cDNA in the fragmentlibrary illustratively comprises digesting the fragment library with atype IIS restriction endonuclease, thereby creating the central site,and ligating the antisense gene DNA at the central site. An illustrativeantisense gene comprises a ribozyme catalytic core, typically, ahammerhead ribozyme catalytic core.

[0021] Another aspect of the invention relates to a method forgenerating a library of DNA fragments of a selected size wherein thefragments collectively span all possible sites of the selected size in asource DNA comprising a first end, a second end, and a central sitethereof, comprising:

[0022] (a) cloning the source DNA in a cloning vector;

[0023] (b) creating a plurality of deletion derivatives of the clonedsource DNA wherein each of the plurality of deletion derivatives has adeletion extending from the first end into the cloned DNA such that theplurality of deletion derivatives comprises a deletion librarycomprising deletions extend serially into the cloned DNA; and

[0024] (c) reducing the size of the DNA contained in the deletionlibrary to a preselected size by removing a portion of the DNA from thesecond end thereof to result in the library of fragments.

[0025] Still another aspect of the invention relates to a method foridentifying target sites for antisense-mediated inhibition of a selectedgene comprising:

[0026] (a) constructing a directed antisense library targeted at theselected gene wherein the library is contained in a cloning vectorhaving a promoter configured for transcribing antisense transcripts fromthe directed antisense library in suitable cells wherein the selectedgene is expressed as a target transcript;

[0027] (b) transforming a plurality of the suitable cells such that eachof the plurality of suitable cells transcribes an antisense transcriptthat has access to the target transcript for potential inactivationthereof;

[0028] (c) identifying a cell wherein an antisense transcriptinactivates the target transcript; and

[0029] (d) analyzing the antisense transcript that inactivates thetarget transcript and determining a target site on the antisensetranscript that is associated with inactivation of the targettranscript.

[0030] Yet another aspect of the invention relates to a method foridentifying target sites for antisense-mediated inhibition of a selectedgene comprising:

[0031] (a) constructing a directed antisense library targeted at theselected gene wherein the library is contained in a cloning vectorhaving a promoter configured for transcribing antisense transcripts fromthe directed antisense library in vitro;

[0032] (b) transcribing antisense transcripts from the directedantisense library in vitro;

[0033] (c) incubating the antisense transcripts with a lysate from acell containing target transcripts transcribed from the selected genesuch that antisense transcripts targeted to the target transcripts bindto such target transcripts; and

[0034] (d) analyzing the antisense transcripts that bind the targettranscript and determining a target site on the antisense transcriptthat is associated with binding of the target transcript.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0035]FIGS. 1A and 1B show illustrative multi-cloning sequences (MCS's)according to an aspect of the present invention.

[0036]FIG. 2 summarizes an illustrative method for making a DNA fragmentlibrary containing 14 bp fragments according to the present invention.

[0037]FIG. 3 shows a schematic representation of a hammerhead ribozymebound to a target substrate; the hammerhead ribozyme comprises acatalytic core that cleaves the substrate at the cleavage site indicatedby the arrow and a recognition domain for binding to the substrate bybase pairing.

[0038]FIG. 4 summarizes an illustrative method for making a hammerheadribozyme library from an antisense RNA library according to the presentinvention.

[0039]FIG. 5A shows an illustrative method for inserting a selectedcassette at an end of a deletion fragment in a deletion fragment libraryaccording to the present invention.

[0040]FIG. 5B shows an illustrative method for inserting a selectedcassette in a MCS prior to preparation of a deletion fragment libraryaccording to the present invention.

[0041]FIG. 6A shows a map of expression vector pBK, which is suitablefor use in identifying antisense targets in mammalian cells according tothe present invention.

[0042]FIG. 6B shows base pairing of nucleotides in a multi-cloningsequence flanked by cis-acting ribozymes (CAR's).

[0043]FIG. 7A shows a map of vector pASlib according to the presentinvention.

[0044]FIG. 7B shows a map of vector pShuttle according to the presentinvention.

[0045]FIG. 7C shows a map of the MCS of pShuttle according to thepresent invention.

[0046]FIG. 8 shows a histogram of the distribution of 56 target sites inan illustrative antisense library according to the present invention.

DETAILED DESCRIPTION

[0047] Before the present compositions and methods for generatingdirected antisense libraries and methods of use thereof are disclosedand described, it is to be understood that this invention is not limitedto the particular configurations, process steps, and materials disclosedherein as such configurations, process steps, and materials may varysomewhat. It is also to be understood that the terminology employedherein is used for the purpose of describing particular embodiments onlyand is not intended to be limiting since the scope of the presentinvention will be limited only by the appended claims and equivalentsthereof.

[0048] The publications and other reference materials referred to hereinto describe the background of the invention and to provide additionaldetail regarding its practice are hereby incorporated by reference. Thereferences discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention.

[0049] It must be noted that, as used in this specification and theappended claims, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise.

[0050] In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outherein.

[0051] As used herein, “antisense agent” and similar terms meanantisense RNA, antisense DNA, and ribozymes.

[0052] As used herein, “comprising,” “including,” “containing,”“characterized by,” and grammatical equivalents thereof are inclusive oropen-ended terms that do not exclude additional, unrecited elements ormethod steps. “Comprising” is to be interpreted as including the morerestrictive terms “consisting of” and “consisting essentially of.”

[0053] As used herein, “consisting of” and grammatical equivalentsthereof exclude any element, step, or ingredient not specified in theclaim.

[0054] As used herein, “consisting essentially of” and grammaticalequivalents thereof limit the scope of a claim to the specifiedmaterials or steps and those that do not materially affect the basic andnovel characteristic or characteristics of the claimed invention.

[0055] Construction of Directed Antisense Libraries

[0056] The present invention includes a procedure that allowsconstruction of directed antisense libraries of a variety of types. Thisrequires the use of specially designed bacterial and/or mammalianplasmid vectors. Most importantly, these vectors possess a speciallydesigned multi-cloning sequence (MCS). This approach is not restrictedto a single MCS, as many can be designed that allow the procedure to beperformed. Two illustrative MCS's are shown in FIGS. 1A (SEQ ID NO:1)and 1B (SEQ ID NO:2 and SEQ ID NO:3). These simply illustrate twopossible multi-cloning sequences that could be used for this method.While some of the same restriction enzyme sites are used in both ofthese MCS's, such particular sites are not necessarily the only sitesthat could be used. Many other restriction enzyme sites could substitutefor any of the restriction sites, allowing the same procedure to beperformed.

[0057] The procedure uses a special multi-cloning sequence and a seriesof enzymatic manipulations to produce DNA fragment libraries directedagainst any desired gene of interest. The fragment libraries contain alloverlapping fragments spanning the entire length of the gene ofinterest. Transcription in vitro or in vivo of the DNA fragment allowsthe production of an antisense RNA targeted to the site on the RNAtranscript that is encoded by the DNA fragment. Transcription of theentire DNA fragment library produces all antisense RNA moleculestargeting all positions on the RNA target. Expression of the library inmammalian cells allows identification of effective target sites forantisense-mediated gene inhibition.

[0058] The procedure is illustrated in FIG. 2 using the MCS shown inFIG. 1A. Beginning with the MCS 10 in a suitable circular plasmid vector(described in more detail below), a blunt-ended DNA fragment encodingthe gene of interest 14 is ligated into the EcoRV-digested MCS (FIG. 2,step a). Since the gene can be inserted in one of two orientations, aclone is selected, according to methods well known in the in art such asnucleotide sequencing or restriction mapping, wherein the gene insert issuitably oriented. The orientation will depend on the placement of atranscriptional promoter adjacent to the MCS. The orientation of theinsert will be chosen such that the antisense strand of the insert willbe transcribed by the adjacent promoter. Next, a deletion library isprepared. The plasmid containing the gene of interest is digested withboth PmeI and BbeI (FIG. 2, step b). The BbeI terminus is protected fromexonuclease III digestion because of its 3′overhang 18, while the PmeIterminus 22 is a suitable substrate therefor. The digested plasmid isthen treated with exonuclease III and aliquots are removed over timeinto a stop mixture (FIG. 2, step c). The time points are chosen suchthat deletions are generated after every nucleotide across the entiregene. After exonuclease III digestion, the combined aliquots are treatedwith mung bean nuclease to remove the resulting 5′ overhang (FIG. 2,step c). The termini are then polished with T4 DNA polymerase (FIG. 2,step d) and the plasmid is re-circularized with T4 DNA ligase to producethe deletion library (FIG. 2, step e). The deletion library is thenconverted into a fragment library (14 base-pair fragments 26 in thiscase) by digestion with restriction endonucleases BsmI and BpmI (FIG. 2,step f), purification of the plasmid containing the 14 bp fragment 26from the excised BpmI/BsmI fragment 30 (FIG. 2, step g), end-polishingwith T4 DNA polymerase (FIG. 2, step h), and ligation with T4 DNA ligase(FIG. 2, step i). Not stated, but implied, after each ligation step(i.e., steps a, e, and i) the ligation mixture is transformed intobacteria, the DNA is recovered from the bacteria, and the recovered DNAis used in the subsequent step. All of these reactions involvingrestriction endonucleases, ligases, polymerases, nucleases, and the likeare well known in the art and are performed according to standardmethods, e.g., J. Sambrook et al., Molecular Cloning: A LaboratoryManual (2d ed., 1989); T. Maniatis et al., Molecular Cloning: ALaboratory Manual (1982); F. Ausubel et al., Current Protocols inMolecular Biology (1987), relevant parts of which are herebyincorporated by reference.

[0059] The essence of the procedure is as follows. A gene of interest isconverted into a library of fragments serially deleted after everynucleotide. This deletion library is subsequently converted into afragment library containing all overlapping fragments encoded by thegene.

[0060] The fragment library can also serve as the starting point forconstruction of other types of antisense libraries. One such library isan antisense hammerhead ribozyme library.

[0061] A hammerhead ribozyme 34 is a small RNA that can catalyze thecleavage of a complementary RNA target 38 (FIG. 3). The hammerheadcomprises a catalytic core 42 (SEQ ID NO:4), essential for cleavageactivity. Additionally, the hammerhead has a recognition domain 46 thatis required for interaction with a complementary substrate, such as anRNA transcript. There are few sequence requirements for the recognitiondomain, thus by changing the sequence of the recognition domain almostany sequence can be targeted for cleavage by the hammerhead. Cleavage ofthe substrate 38 occurs at a cleavage site 50 containing an NUH sequence(where N is A, C, G, or U and H is A, C, or U). In the case where thesubstrate is a gene transcript, the hammerhead can be used as anantisense inhibitor of gene expression.

[0062] To convert the fragment (antisense RNA) library into a hammerheadribozyme library, a DNA fragment encoding the hammerhead catalytic coreis inserted into the DNA fragment encoding the antisense RNA. This isperformed as illustrated in FIG. 4. The 14 base-pair fragment 54 in thefragment library is bisected with HphI (FIG. 4, step a). The resultingsingle-stranded overhang on each terminus is then removed using the 3′to 5′ exonuclease activity of T4 DNA polymerase (FIG. 4, step b) toresult in blunt ends 58, 62. A DNA fragment encoding the hammerheadcatalytic core 66 (SEQ ID NO:4) is then inserted by ligation (FIG. 4,step c). The catalytic core shown in FIG. 4 is interrupted by apromoter-less chloramphenicol resistance gene 70 (CAT). A promoter isprovided flanking the MCS. Transforming bacteria and selecting forchloramphenicol resistance allows selection for clones in which thecatalytic core is in the correct orientation to produce a bonafidehammerhead ribozyme. Next, the CAT gene is removed and the sequenceencoding a hammerhead ribozyme 74 is generated by NruI digestion (FIG.4, step d) and ligation with T4 DNA ligase (FIG. 4, step e).

[0063] Other types of antisense libraries can also be produced from thefragment library. For instance, other cassettes can be ligated into anHphI-digested fragment library. Catalytic cores from other ribozymes,including those currently known and those to be discovered, can beinserted. Additionally, other cassettes could be used that encodesequences that cause modification to the target by mechanisms other thancleavage. Similarly, ribozyme and non-ribozyme sequences can be added tothe end of the antisense sequence. This is illustrated in FIG. 5A,wherein the DNA fragment library is digested with BpmI, which digeststhe DNA at the distal end of the inserted fragment 78 (step a). Theunpaired nucleotides resulting from this reaction are then removed withT4 DNA polymerase (step b) to result in blunt ends 82, 86. Next, acassette 90 is inserted by ligation to recircularize the modifiedplasmid 94, now containing the cassette inserted at an end of the insertfragment. Alternatively, instead of inserting a cassette after thefragment library is produced, a suitable cassette can be engineered intothe starting multi-cloning sequence. For instance, the HphI site of theoriginal MCS (FIG. 1A) could be replaced with a cassette encoding anydesired sequence (FIG. 5B). Then, using the same procedure illustratedin FIG. 2, the cassette can be placed against the fragment sequence inthe conversion of the deletion library into the fragment library. Anexample of a possible cassette is one encoding the sequence CUGA. Anantisense RNA with this sequence at its 3′ end has been shown to becapable of directing the 2′-O-methylation of the complementary target(J. Cavaille et al., Targeted ribose methylation of RNA in vivo directedby tailored antisense RNA guides, 383 Nature 732-735 (1996)). Thisreaction is catalyzed by modification machinery present in mammaliancells. 2′-O-methylation of a suitable target site could be used toinhibit expression of the RNA transcript. Other cellular RNA processingreactions can also be used in a similar fashion with the use ofdifferent cassettes placed adjacent to the antisense RNA sequence.

[0064] Use of Directed Libraries in the Identification of Target Sitesfor Antisense-Mediated Gene Inhibition

[0065] Antisense libraries prepared according to the present inventioncan be assayed in vitro in a cell free system or in vivo in culturedcells, as will be described in more detail below.

[0066] In vivo assay. For in vivo, use the antisense library isintroduced by transfection into a suitable cell line that expresses thegene of interest. The transfection conditions are chosen such that onlyone member of the library is taken up by each individual cell. Theindividual cells then each express a different antisense moleculetargeted to a different site on the RNA transcript of interest. Alltarget sites are represented in the entire cell population produced bytransfection. Using a suitable detection method, cell clones can beidentified in which expression of the target RNA has been reduced oreliminated. These clones possess an antisense molecule that targets aneffective site on the RNA transcript of interest. The plasmid encodingthis antisense molecule is recovered and the target sequence isidentified by DNA sequencing.

[0067] To identify suitable targets in vivo, specially designedexpression vectors are required. One key feature of such expressionvectors is that they are designed to replicate episomally in mammaliancells. FIGS. 6A and 7B show two such episomal vectors, pBK (SEQ ID NO:17) and pShuttle (SEQ ID NO: 14), respectively. Vector pBK possesses theorigin of replication and the gene encoding the T/t antigen from thehuman papova virus BK (BKV). Vector pShuttle possesses the origin ofreplication and the EBNA1 gene from the human Epstein-Barr virus (EBV).These sequence elements allow each of the plasmids to replicateextrachromosomally (episomally). Episomal expression is desirable forseveral reasons. First, it eliminates the clone-to-clone variation inexpression that occurs if stable transfectants are used. P. B. Belt etal., 84 Gene 407-417 (1989). Second, since the copy number of theepisomal vector is determined primarily by the transfection conditionsand, once established, remains tightly regulated, J. L. Yates & N. Guan,65 J. Virol. 483-488 (1991), then effects on expression due todifferences in copy number are minimal. Consequently, the selection ofantisense efficacy is based on accessibility and not the level ofexpression. Third, the use of an episomal expression vector allows forhigh transfection efficiency. P. B. Belt et al., 84 Gene 407-417 (1989);R. F. Maragolskee et al., 8 Mol. Cell. Biol. 2837-2847 (1988). This isimportant to ensure that all antisense agents present in the library arerepresented in the mammalian transfectants. Finally, the plasmid can berecovered and shuttled back into bacterial cells. This allows thesequence of effective antisense agents to be determined, therebyidentifying accessible target sites. As a demonstration of episomalreplication, pShuttle was used to transfect HeLa cells, and the cellswere grown in culture under 400 μg/ml hygromycin selection. After 1month in culture, low molecular weight DNA was isolated from 1×10⁷ cellsand used to transform Escherichia coli DH5α, producing a total of 2475hygromycin-resistant colonies.

[0068] Vector pBK illustrates other features of value for in vivoexpression of antisense libraries. pBK has a single antibioticresistance gene, bleomycin^(R), driven by dual mammalian (CMV) andbacterial (em7) promoters. This allows the same selectable marker to beused in both bacterial and mammalian cells. This helps to minimize thesize of the vector, since large vectors transfect at a lower efficiency.pBK has both the BK origin of replication and the origin of replicationfrom the pUC series of bacterial plasmids. Therefore pBK can bereplicated in both bacterial and mammalian cells, and can be shuttledbetween them. pBK was designed such that the antisense library could beconstructed and expressed from the same vector. The antisense sequenceis expressed by read-through expression of the bleomycin^(R) gene. Thisensures expression of the antisense agent when the cells are grown inthe presence of bleomycin. The antisense fragment is released from thelarger bleomycin transcript by the activity of cis-acting ribozymes(CAR), hammerhead ribozymes in this case, that flank the antisensesequence. In the absence of CAR, flanking sequences of the largerbleomycin transcript could inhibit the activity of the antisense agent.Sequences outside of the MCS (FIG. 1A) encode the cis-acting ribozymes.They are illustrated in FIG. 6B where only the sequence of the upperstrand of the MCS is shown (SEQ ID NO:18). On cleavage by the CAR, theantisense agent is released and stable hairpin loops form to increasethe nuclease resistance of the antisense agent.

[0069] pShuttle shares many of the same features as pBK, with twosignificant differences. First, this episomal vector is EBV-based ratherthan BKV-based. The second and more significant difference is thatconstruction of the antisense library is not possible in pShuttle.Instead, the antisense library is first constructed in pASlib (SEQ IDNO:7), and subsequently transferred to pShuttle for expression inmammalian cells. The antisense encoding fragment of pASlib is removed bydigestion with HindIII and SalI (FIG. 7A). Subsequently, theHindIII/SalI fragment is ligated into the multi-cloning site of pShuttlevia the HindIII and XhoI sites (FIG. 7C). This places the antisensesequence downstream of a dual CMV/T7 promoter for expression in vivo inmammalian cells or, alternatively, in vitro by transcription using T7RNA polymerase.

[0070] Although it is believed that episomal shuttle vectors areadvantageous for expression of directed antisense libraries, viralvectors can also be used. Many viruses are currently being examined forexpression of foreign genes for the purpose of gene therapy. These sameviral vectors would be suitable for expression of directed antisenselibraries. Some of these vectors replicate extrachromosomally andtherefore behave similarly to the described episomal vectors. Othersintegrate into chromosomes. For the use of integrative viral vectors,two minor problems would need to be dealt with. First, the antisensegene present within the viral vector would integrate into the chromosomewith the virus. Consequently, recovering the gene to determine the siteat which it targets is not readily possible. This can be dealt with byusing polymerase chain reaction (PCR) to amplify the integratedantisense gene. The PCR product could be sequenced directly, or clonedand sequenced to identify the target site. Second, some of these viralvectors integrate randomly and this would produce differing levels ofexpression from different members of the directed antisense library. Asdiscussed, it is important that expression of all members of the librarybe comparable. This problem can be dealt with by using a viral vectorthat integrates at a specific preferred site, such as adeno-associatedvirus.

[0071] In vitro assay. Identification of effective antisense targetsites using antisense libraries can also be performed using in vitroassays. For instance, an assay such as that used by Lieber and Strauss(A. Lieber & M. Strauss, Selection of efficient cleavage sites in targetRNAs by using a ribozyme expression library, 15 Molecular and CellularBiology 540-551 (1995)), can be used. For this, the antisense library isproduced by in vitro transcription from a suitable promoter. In thepresent case, an antisense ribozyme library in pShuttle might be used.Of course other types of antisense libraries could be used similarly.The library-containing pShuttle is digested with XbaI and used as atemplate for run-off transcription of the antisense ribozyme by in vitrotranscription with T7 RNA polymerase, according to methods well known inthe art (e.g., C. J. Noren et al., 18 Nucleic Acids Res. 83-88 (1990).Subsequently, the transcribed ribozyme library is incubated in a lysateprepared from a mammalian cell line expressing the gene of interest.Effective target sites are identified by performing a primer extensionreaction on purified RNA from the lysate using a primer specific for thegene of interest. Primer extension products terminate at the sites ofcleavage by effective ribozymes. These sites are identified by gelelectrophoresis of the primer extension products with suitable sizemarkers.

EXAMPLE 1

[0072] Construction of pASlib. In this example, there is described anillustrative plasmid according to the present invention for making adeletion library of a selected DNA. This plasmid was constructed asfollows.

[0073] The HindIII-HpaI fragment of pLA2917 (J. N. Allen & R. S. Hanson,161 J. Bact. 955-962 (1985)), containing the kanamycin resistance gene,was inserted into HindIII/SmaI-digested pUC19 to produce pUCKan. An HphIand two BsaHI sites were eliminated from the kanamycin resistance geneby site-directed mutagenesis, according to methods well known in theart, to produce pUCKan*. The mutagenized kanamycin resistance gene wasremoved by HindIII/EcoRI digestion, and the termini were blunted by5′-overhang fill-in using the Klenow fragment of DNA polymerase I andligated to the 843 bp BspHI-SapI fragment of pUC 19 containing theorigin of replication. A clone (pKan) was selected wherein the EcoRI andBspHI sites were juxtaposed. The BsmFI and PstI sites were eliminatedfrom pKan by site-directed mutagenesis using the procedure of E. Merinoet al., 12 Biotechniques 508-510 (1992). The multiple cloning site forpASlib was constructed from the overlapping oligodeoxynucleotides MCS-L(SEQ ID NO:5) and MCS-R (SEQ ID NO:6) by 5′-overhang fill-in with theKlenow fragment of DNA polymerase I. Oligonucleotides were synthesizedusing an Applied Biosystems automated oligonucleotide synthesizer. Thedouble-stranded multiple cloning site was inserted into EcoRI-linearizedand blunted pKan to result in pASlib (SEQ ID NO:7). Restrictionendonuclease digestions, primer extension reactions, ligation reactions,and the like were carried out according to methods well known in theart. E.g., J. Sambrook et al., Molecular Cloning: A Laboratory Manual(2d ed., 1989); T. Maniatis et al., Molecular Cloning: A LaboratoryManual (1982); F. Ausubel et al., Current Protocols in Molecular Biology(1987).

[0074] Therefore, pASlib possesses the pUC19 origin of replication and akanamycin resistance gene allowing selection in bacterial cells. Thekanamycin resistance gene was chosen as the selectable marker since, ofall the available bacterial selection markers, it possessed the fewestsites present in the MCS. Therefore, it was the simplest to modify bysite-specific mutagenesis to eliminate the undesirable sites. The MCScontains the following salient features. It possesses a short polylinkerthat allows much flexibility in the cloning of the gene or cDNA sequenceof interest, which represents the first step in the construction of anantisense library. The polylinker includes several restriction sitesthat leave sticky ends upon digestion. These sites can be used todirectionally clone the cDNA or genomic fragment in the correctorientation. Alternatively, the fragment can be cloned by blunt-endligation, and the correctly oriented clone can be selected byrestriction analysis. The PstI and PmeI sites allow the generation of asubstrate for unidirectional digestion by exonuclease III into thecloned cDNA or genomic fragment. This allows preparation of a serialdeletion library of the cloned insert. The BsmFI and BbsI sites are usedtogether to convert the deletion library into a 14 bp fragment library.The HphI site allows bisection of the 14 bp fragment library forintroduction of the antisense agent.

EXAMPLE 2

[0075] Construction of pShuttle. In this example, there is described theconstruction of an illustrative plasmid for use according to the presentinvention for expressing an antisense agent in either mammalian cellsusing the intermediate-early promoter from cytomegalovirus or in vitrousing T7 polymerase.

[0076] A hygromycin expression cassette capable of being expressed inboth mammalian and prokaryotic systems was constructed using overlapextension PCR. PCR was carried out according to methods well known inthe art, e.g., U.S. Pat. No. 4,683,195; U.S. Pat. No. 4,683,202; U.S.Pat. No. 4,800,159; U.S. Pat. No. 4,965,188; PCR Technology: Principlesand Applications for DNA Amplification (H. Erlich ed., Stockton Press,New York, 1989); PCR Protocols: A guide to Methods and Applications(Innis et al. eds, Academic Press, San Diego, Calif., 1990). The 1026 bphygromycin resistance coding sequence from EBOpLPP (ATCC) was joined atits 3′-end to the 322 bp 3′-untranslated region (UTR)/SV40 earlypolyadenylation sequence from pRC/CMV (Invitrogen Corp., Carlsbad,Calif.), while the 527 bp dual ampicillin/SV40 early promoter frompEGFP-1 was joined to the 5′-end. The sequences of the primers used inthe PCR were as follows: 3′-UTR/poly(A) segment, SEQ ID NO:8 and SEQ IDNO:9; hygromycin coding region, SEQ ID NO: 10 and SEQ ID NO: 11;amp/SV40 early promoter, SEQ ID NO: 12 and SEQ ID NO: 13. Each portionof the hygromycin cassette was prepared by PCR using one of the threeprimer sets and the appropriate template. The resulting fragments weregel purified. The hygromycin-encoding and the 3′-UTR/poly(A) fragmentswere combined and used in a second PCR reaction to produce thehygromycin-3′-UTR/poly(A) fragment. In a final PCR, this fragment wascombined with the amp/SV40 fragment to produce the complete 1875 bphygromycin gene cassette. The hygromycin gene cassette was ligated intothe 843 bp BspHI-SapI oriP-containing fragment of pUCl9, producing pHyg.The 4914 bp EcoRI-BamHI fragment containing the EBNA-1 and EBV oriPsequences from EBOpLPP was inserted between the hygromycin cassette andthe pUC 19 origin of XhoI-digested pHyg to make pEBV. The 1060 bpexpression cassette was excised from pRC/CMV using NruI and PvuII andinserted into the BamHI site of pEBV to produce pShuttle (SEQ ID NO:14).

[0077] pShuttle was designed to allow replication and expression of theantisense library in mammalian cells. It possesses an MCS for insertionof the antisense library. The MCS is flanked on one end by a dual CMV/T7promoter for allowing expression of the antisense agent gene both inmammalian cells as well as by in vitro transcription using T7 RNApolymerase. On the other end of the MCS is a bovine growth hormonepolyadenylation signal for efficient expression in mammalian cells.pShuttle possesses a hygromycin resistance gene driven by a dualpromoter for allowing selection in bacterial and mammalian cells. ThepUC 19 origin of replication allows replication in bacterial cells. Forreplication in mammalian cells, the EBV origin and EBNA-1 gene wereincluded. J. Yates et al., 81 Proc. Nat'l Acad. Sci. USA 3806-3810(1984); J. Yates et al., 313 Nature 812-815 (1985).

EXAMPLE 3

[0078] Construction of a hammerhead ribozyme catalytic core cassette. Acassette encoding the hammerhead catalytic core, interrupted by the CATgene (S. Horinouchi & B. Weisblum, 150 J. Bact. 815-825 (1982)), wasconstructed as follows. PCR primers were prepared that werecomplementary to the CAT gene on their 3′-ends and encoded thehammerhead catalytic core on their 5′-ends. The sequences of the primerswere SEQ ID NO: 15 and SEQ ID NO:16. Located between the CAT andhammerhead catalytic core sequences were NruI restriction sites. The PCRcontained 5 ng CAT gene DNA, 100 pmol each of the primers CatCass 1 andCatCass 2, 1 mM of each of the four dNTPs, 5 units of VENT polymerase(New England Biolabs, Beverly, Mass.) in the standard VENT polymerasebuffer except that the concentration of the MgSO₄ was increased to 5.2mM (i.e., 10 mM KCl, 10 mM (NH4)₂SO_(4, 20) mM Tris-HCl (pH 8.8 at 24°C.), 5.2 mM MgSO₄, 0.1% Triton X-100). The use of VENT polymeraseensured that the cassette possessed blunt ends.

[0079] The reaction mixture was incubated as follows:

[0080] (A) 2 minutes at 94° C.;

[0081] (B) 5 cycles of 1 minute at 94° C., 30 seconds at 45° C., and 2minutes at 72° C.;

[0082] (C) 15 cycles of 30 seconds at 94° C., 15 seconds at 60° C., and2 minutes at 72° C.; and

[0083] (D) 5 minutes at 73° C.

[0084] After amplification, the cassette was purified fromunincorporated primers by agarose gel electrophoresis, and the agarosewas subsequently removed from the cassette DNA using standardprocedures.

[0085] Introduction of a catalytic core into a fragment library presentsseveral difficulties. The core must be inserted by blunt-end ligationand in the correct orientation to produce a functional ribozyme.Additionally, due to its small size, it is difficult to prevent theintroduction of concatamers of the core and/or contamination of thelibrary with clones that do not acquire a catalytic core. To increasethe effectiveness and efficiency of this step, the core interrupted bythe CAT gene was designed. CAT selection allows the use of anon-phosphorylated cassette. This prevents insertion of multimers andselects against non-recombinants. Additionally, the CAT gene allowsselection of clones acquiring a correctly oriented catalytic core. Inthe desired orientation, transcription of the CAT and kanamycin genes isin the same direction. In the incorrect orientation, CAT expression isinhibited by antisense expression from the kanamycin resistance gene.This phenomenon has been noted previously, R. Bruckner et al., 32 Gene151-1160 (1984). After selection, the CAT gene is removed by digestionwith NruI to produce a sequence encoding a hammerhead ribozyme.

EXAMPLE 4

[0086] Construction of a Herpes ICP4 ribozyme library. The 4489 bpBglII-EcoRI fragment from pTEG2, X. X. Zhu et al., 184 Virology 67-78(1991), containing a herpes simplex virus ICP4 genomic fragment wascloned into EcoRI-EcoRV-digested pASlib. This fragment included 125 bpupstream of the translational start site, 466 bp downstream of thetranslational termination sequence, and the entire genomic codingsequence of ICP4. The resulting clone, pASlib-ICP4, contained the ICP4fragment with the sense strand as the upper strand.

[0087] From pASlib-ICP4 a deletion library was produced as follows.Twenty μg of CsCl gradient purified plasmid DNA was digested with PstIand XbaI, then concentrated and desalted using a Microcon 50 spin filter(Amicon). The DNA was brought up to a volume of 60.4 μl of exonucleaseIII buffer (i.e., 50 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 10 mM2-mercaptoethanol), warmed to 37° C., and then 300 units of exonucleaseIII were added. At 1-minute intervals after the addition of theexonuclease III, 2.5 μl of the reaction mixture was removed and placedin microfuge tubes on ice containing 7.5 μl of 66.7 mM sodium acetate,pH 5.2, 200 mM NaCl, 1.3 mM ZnCl₂, and 1 unit of mung bean nuclease.After 25 aliquots had been removed, the mung bean nuclease-containingtubes were incubated at 20° C. for 30 minutes. After 30 minutes, thecontents of all of the mung bean nuclease-containing tubes were combinedand extracted with phenol-chloroform (50:50), extracted with chloroform,and then were precipitated with two volumes of 100% ethanol. The DNA wasthen pelleted and dried. The DNA was then resuspended in 18 μl of d.i.H₂O, 2.5 μl of 10 mM of each of the four dNTPs, 2.5 μl of 10×Pfupolymerase buffer (e.g., 100 mM KCl, 100 mM (NH₄)₂SO₄, 200 mM Tris-Cl(pH 8.75), 20 mM MgSO₄, 1% Triton® X-100, 1000 mg/ml BSA), and 5 unitsof Pfu polymerase (Stratagene, La Jolla, Calif.), then incubated at 72°C. for 15 minutes, and cooled to room temperature. The plasmid DNA wasrecircularized by ligating in a large volume (1.25 ml) in a buffercontaining 5% PEG for 4 hours at room temperature. Except for themodifications indicated, all ligations were performed with T4 DNA ligaseunder the conditions suggested by the manufacturer.

[0088] After transformation into E. coli DH5 a, the cells were grown inliquid culture to amplify the deletion library. The library DNA waspurified and digested with BsmFI and BbsI, and then the ends wereblunted with Pfu polymerase as described above. The DNA was thenrecircularized by ligation in a 600 μl volume in a buffer containing 5%polyethylene glycol (PEG) at room temperature for 4 hours. Aftertransformation into E. coli DH5α, amplification and plasmidpurification, 1 μg of library plasmid DNA was then subjected todigestion with 8 units of HphI for 1 hour at 37° C., and the ends werepolished with T4 DNA polymerase. The hammerhead core cassette wasinserted by ligating 0.5 μg of the HphI-digested library DNA with 5 μgof the ribozyme core sequence cassette prepared according to theprocedure of Example 3. That ligation product was transformed into DH5αand grown in culture under chloramphenicol selection. Afterpurification, 2 μg of the DNA was digested with HindII and SalI, and theterminal phosphates were removed using shrimp alkaline phosphatase(Amersham, Arlington Heights, Ill.). The HindIII/SalI digest wasfractionated on an agarose gel, and the dephosphorylatedribozyme/chloramphenicol cassette was purified using standardprocedures. The cassette was combined with an equimolar amount ofHindIII-XhoI-digested pShuttle, prepared according to the procedure ofExample 2, and ligated using a modified two-step ligation procedure, S.Damak & D. W. Bullock, 15 Biotechniques 448-450 (1993) (herebyincorporated by reference). The first step was performed at roomtemperature for 1 hour, and the second step was incubated overnight at16° C. The ligation mixture was transformed in DH5α and grown in cultureunder chloramphenicol selection. The library DNA was purified and thendigested with NruI to release the chloramphenicol gene. The digested DNAwas then recircularized by ligation in a volume of 600 μl. The finalligation product was transformed into DH5α, and the plasmid DNA waspurified on a CsCl gradient.

[0089] To verify the effectiveness of this procedure, 56 clones obtainedat various steps were sequenced. Thirty-one were from the final ribozymelibrary, and the remainder were from earlier steps, beginning with the14 bp fragment library. The results of the sequencing are discussedbelow.

[0090] One observation made after the mung bean digestion was that thedeletions infrequently stopped at A-T base pairs. While exonuclease IIIhas been shown to exhibit a preference for stopping at certainnucleotides (C>A=T>G), W. Linxweiler & W. Horz, 10 Nucleic Acids Res.4845-4859 (1982), this was not believed to be the cause of the observedsequence bias. Instead, it is believed that this is the result of agreater degree of “breathing” at A-T terminated deletions and thesubsequent removal of A-T terminal pairs by mung bean nuclease. The mungbean nuclease digestion was later performed at higher saltconcentrations (150 mM) and at a lower temperature (20° C.). Thiseliminated the under-representation of A-T terminated deletions.

[0091] For construction of the library, two type IIS restriction enzymesare required, BsmFI and HphI. Typical of type IIS restriction enzymes,BsmFI and HphI cleave downstream of their recognition sequences in asequence-independent manner. Cleavage by type IIS restriction enzymescan pose some problems since they can exhibit infidelity in how far fromtheir recognition site they cleave. Cleavage by BsmFI was largely at theexpected distance (10/14), but also at 11/15. The reported 9/13 activityfor this enzyme, V. E. Velculescu et al., 270 Science 484-487 (1995),was not seen in any of the clones sequenced. Infidelity of BsmFI doesnot present a problem for construction of ribozyme libraries. The resultof this infidelity is that the recognition domains of the ribozymes inthe library can vary from 13 to 15 nucleotides.

[0092] In contrast, HphI infidelity can be problematical. HphI digestionis a critical step in the construction of ribozyme libraries. Thisenzyme produces a 1 nucleotide 3′-overhang that is later removed bypolishing with T4 DNA polymerase. It is essential to the properfunctioning of the resulting ribozyme that this 1 nucleotide be removed,since it does not have an antisense binding partner in the ribozyme(FIG. 1, X).

[0093] HphI cleaves at 8/7, but also at 9/8. D. Kleid et al., 73 Proc.Nat'l Acad. Sci. USA 293-297 (1976). This infidelity is demonstrated inthe present library by the presence of ribozymes with flanking helicesof length 8 and 5, as would be expected if HphI cleaved at 9/8. Thistype of infidelity, in itself, is not problematical. It simply altersthe relative lengths of the two arms of the binding domain, leaving thetotal length of the binding arms unchanged. The problem that arises withHphI infidelity is that the enzyme can cleave twice at the same target,i.e., if it first cleaves at 9/8 it can rebind and cleave at 8/7. Theresult is that 2 bp are removed from the sequence upon polishing with T4DNA polymerase. Removal of 2 bp from the insertion site of the ribozymecore produces a non-functional ribozyme. In early attempts to produce alibrary, >40% of the clones were the product of double cutting. This isclose to the statistically predicted 50% that would occur if HphI has nopreference for either 8/7 or 9/8 cutting. To minimize the possibility ofdouble cutting, HphI digestion was performed under near “single hit”conditions. Under these conditions, double cleavage was only 13% of thefinal library. It should be possible to further reduce the percentage ofdouble hits by performing the cleavage under “sub-single hit”conditions. This should not present any problem so long as the amount ofplasmid digested is sufficient to allow full representation of theribozyme library. Undigested molecules cannot accept the catalytic coreand are removed in the later step by selection for chloramphenicolresistance. Other class IIS restriction enzymes, such as MboII, couldlikely substitute for HphI, but fidelity may not be any better.

[0094] The infidelity of HphI raises another issue. It is possible thatsome sequences favor digestion at 8/7 and others at 9/8. This could leadto the absence of some ribozyme target sequences in the final library.This appears to be unlikely, however. First, as discussed, underconditions that give nearly 100% cleavage by HphI, >40% of the moleculesare cut twice. This is close to the 50% predicted if HphI exhibits nopreference for 8/7 versus 9/8 cutting. Second, two clones that bothcontain the same 14 bp sequence of ICP4, Rz8 and Rz9 (Table 1), are theproducts of 8/7 and 9/8 cleavage, respectively. This suggests that theintervening sequence between the binding site and the cleavage site doesnot affect where HphI cleaves.

[0095] HphI is also sensitive to overlapping dam methylation. This isalso true of MboII. Since 2 nucleotides of the four base consensussequence for dam methylation are provided by the variable sequence ofthe cDNA insert, mathematically {fraction (1/16)} of the clones in the14 bp fragment library (6.25%) will not be cleaved with HphI and will beeliminated from the final ribozyme library. This can be prevented bypassage of the 14 bp fragment library in a dam⁻ strain prior to HphIdigestion. TABLE 1 Clone (Position) Target Seguence^(a) SEQ ID NO: Rz1(1754) cgacgccgcccgcc 19 Rz2 (1992) cugcgcgcguggcu 20 Rz3 (2045)gcgccugcgcgggg 21 Rz4 (2252) cgccgccgacgcgc 22 Rz5 (2411) cccccuccccgcg23 Rz6 (2517) guggcccugucgcg 24 Rz7 (2590) gccacacggcggcg 25 Rz8 (2729)cgccgcgcggugcg 26 Rz9 (2729)^(b) cgccgcgcggugcg 27 Rz10 (2837)cccccugcgcgccuc 28 Rz11 (2915) gguggugcuguacuc 29 Rz12 (3246)gggcccgcgguguc 30 Rz13 (3275)^(c) ccuggcgugcgagc 31 Rz14 (3569)ggggaccaccgacgccauggc 32 Rz15 (3680) cguggcgcuggggc 33 Rz16 (3842)cgggauucgcuggg^(d) 34

[0096] The target locations of the 56 sequenced clones are illustratedin FIG. 8. The histogram indicates that the target sites are fairlyevenly distributed across the entire ICP4 gene, with the exception thatno clones were identified targeting the 5′- and 3′-termini. It isunlikely that the library is devoid of members targeting these regionssince the libraries are prepared with complexities far exceeding thetotal number of sites on the gene. It is even possible that target sitesin these regions are similarly represented as those identified by thesequenced clones. Due to the small number of clones sequenced, it islikely that some larger gaps in the data could be observed even for auniformly represented library, such as the gap between positions 966 and1282.

[0097] Of the 56 sequences determined, 42 (75%) occurred only once,while four occurred multiple times (FIG. 8). Three were only mildlyover-represented, with two or three occurrences compared with the singleoccurrence for the majority of clones. The three positions were 2054 and3246, with two occurrences each, and position 2729, with threeoccurrences. One position, 3275, was significantly over-represented,occurring seven times. Five of the occurrences were observed within the32 clones sequenced from the final library, and the other two were foundat early stages of the construction. The over-representation ofparticular sites is likely caused by some local sequence and/orstructure in the DNA that either stalls exonuclease III or causes it tofall off the template. P. Abarzua & K. J. Marians, 81 Proc. Nat'l Acad.Sci. USA 2030-2034 (1984). Performing the exonuclease III deletion athigher temperatures might reduce this phenomenon if an inhibitorystructure is forming at certain sequences. Higher temperature alsoallows for more distributive activity from the enzyme, J. D. Hoheisel,209 Anal. Biochem. 238-246 (1993), which is desirable in this type ofexonuclease III digestion. While it is possible that the exonuclease IIIdigestion conditions may need to be optimized for each target cDNA,creating libraries larger than would be necessary to represent everyposition would ensure complete representation of all target sites.

[0098] Examination of the 31 clones obtained from the final libraryallowed determination of the overall effectiveness of the procedure. All31 possessed a catalytic core, demonstrating the effectiveness of theuse of CAT selection for this purpose. Nineteen of the 31 clones *\(61%)contained sequences that could potentially be ribozymes if the sequencethat they targeted included the required NUH sequence at the correctlocation. These are shown in Table 1. Counted among these potentialribozymes were three clones that possess non-detrimental defects. Onehas a single nucleotide deleted from loop II of the ribozyme (Rz13).This produces a three-, instead of a four-nucleotide loop II. The siteof this defect is the NruI site used to remove CAT from the catalyticcore. The ends must have been damaged during this step for this clone.The other two non-detrimental defects were the result of incompletedigestion by BsmFI. These clones have a longer flanking armcorresponding to helix III (Rz12 and Rz14). This appears to be theresult of a lack of cleavage of the BsmFI site on pASlib and, instead,an internal BsmFI site on ICP4 was used. These clones would be expectedto produce functional ribozymes had they targeted an NUH sequence.

[0099] The remaining 12 clones (39% of 31) possessed defects that wouldprevent them from being potentially functional ribozymes. Four of these(13%) were defective in that they were cleaved twice with HphI. Asdiscussed above, it is likely that this defect can be reduced to closeto zero by performing the HphI digestion under “sub-single hit”conditions. Three (9.7%) were missing 1 nucleotide from one end of thecatalytic core. Since the deletion always occurred at the same end ofthe cassette and the thermostable polymerase used to make the cassettedoes not contain any 5′ to 3′ exonuclease activity, the PCR primerconstituting that end of the cassette must have been contaminated with asmall percentage of a failure fragment of the DNA synthesis. This defectcan be eliminated by better purification of the primers. Five clones(16%) possessed the catalytic core in the incorrect orientation. This isin contrast to the expected 50% if there were no selection fororientation. Incorrectly oriented clones could be eliminated by movingthe promoter for the CAT gene outside the MCS of pASlib. Finally, threeclones were the result of various unknown cloning artifacts.

[0100] Therefore, the success rate of this library was 61%. Asdiscussed, a few procedural changes would increase the success rate to70-80%. This could be increased a further 16% by placing the CATpromoter outside the MCS. Even at 61%, the success rate is more thanadequate. This just means that it is necessary to screen an antisenselibrary 140% the size needed if 100% success were achieved. This wouldstill be a small library relative to a non-directed library approach.

[0101] Three of the 31 clones (9.7%) targeted a site on the ICP4 mRNAthat contained a uridine at the proper position of the consensus NUHsite (Rz3, Rz5, and Rz16). Of the three, only one targeted a consensusNUH site (Rz16). Due to the unusually high G/C content of the ICP4genomic fragment used to make the ribozyme library, only 9.2% of thenucleotides in the mRNA are uridines, of which 203 occur as an NUHtriplet. The fact that the percentage of sequenced clones in the librarytargeting an NU site is virtually identical to the percentage ofuridines in the ICP4 gene suggests that the library is unbiased andlikely to contain a fairly uniform distribution of target sites.

[0102] The use of a direct library for target site selectionsignificantly simplifies the screening process, since only very smalllibraries need be prepared and assayed. For ICP4, assuming the librarycontains a uniform distribution of the 4475 distinct sequences(4489-14), a library of 67,125 (15-fold excess) is expected to have aprobability of 99.9% of containing all sequences. W. Feller, AnIntroduction to Probability Theory and Its Applications (3d ed. 1968).Based on a χ² goodness-of-fit analysis of the 56 sequences, themultiples observed at positions 2729 and 3275 occur with a higherfrequency than would be expected for a uniform distribution. All otherpositions are consistent with a uniform distribution. Correcting for thetwo over-represented sequences, a library of 81,057 (18-fold excess) isexpected to contain all sequences with probability of 99.9%.Preparation, manipulation, and screening of such a library is wellwithin the limitations of current practice. In contrast, a non-directedlibrary targeting 14 nucleotides would require a minimum size of 2.7×10⁸(4¹⁴). The ability to prepare and screen such a library is questionable.Even if possible, the vast majority of members of the library aredirected at non-target genes. Inhibition of non-target genes could poseproblems in interpreting the results.

1 50 1 62 DNA Artificial Sequence Multiple cloning site for use inmaking deletion libraries. 1 gcttggtgat gcattcgata tcgtttaaac gcccgggcgcggccgcggcg cctccagtcg 60 ac 62 2 24 DNA Artificial Sequence Portion of amultiple cloning site for use in making deletion libraries. 2 gtcgacgggactgcaggttt aaac 24 3 23 DNA Artificial Sequence Portion of a multiplecloning site for use in making deletion libraries. 3 gaagacagtcaccaagcttc agc 23 4 23 DNA Unknown Catalytic core of hammerheadribozyme. 4 ctgatgaggt cgcgagaccg aaa 23 5 49 DNA Artificial SequencePCR primer for construction of pASlib. 5 aagcttggtg actgtcttcgagctcgaatt catcgatatc tagagttta 49 6 34 DNA Artificial Sequence PCRprimer for construction of pASlib. 6 gtcgacggga ctgcaggttt aaactctagatatc 34 7 2077 DNA Artificial Sequence pASlib 7 tcagtggaac gaaaactcacgttaagggat tttggtcatg aattgtcgac gggactgcag 60 gtttaaactc tagatatcgatgaattcgag ctcgaagaca gtcaccaagc ttattcccag 120 agtcacgctc agaagaactcgtcaagaagg cgatagaagg cgatgcgctg cgaatcggga 180 gcggcgatac cgtaaagcacgaggaagcgg tcagcccatt cgccgccaag ctcttcagca 240 atatcacggg tagccaacgctatgtcctga tagcggtccg ccacacccag ccggccacag 300 tcgatgaatc cagaaaagcggccattttcc accatgatat tcggcaagca ggcatcgcca 360 tgggtcacga cgagatcctcgccgtcgggc atgcgcgcct tgagcctggc gaacagttcg 420 gctggcgcga gcccctgatgctcttcgtcc agatcatcct gatcgacaag accggcttcc 480 atccgagtac gtgctcgctcgatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc 540 ggatcaagcg tatgcagccgccgcattgca tcagccatga tggatacttt ctcggcagga 600 gcaagatgag atgacaggagatcctgcccc ggcacttcgc ccaatagcag ccaatccctt 660 cccgcttcag tgacaacgtcgagcacagct gcgcaaggaa cgcccgtcgt ggcaagccac 720 gatagccgcg ctgcctcgtcttgcagttca ttcagggcac cggacaggtc ggtcttgaca 780 aaaagaaccg gccgcccctgcgctgacagc cggaacacgg cggcatcaga ggagccgatt 840 gtctgttgtg cccagtcatagccgaatagc ctctccaccc aagcggccgg agaacctgcg 900 tgcaatccat cttgttcaatcatgcgaaac gatcctcatc ctgtctcttg atcagatctt 960 gatcccctgc gccatcagatccttggcggc aagaaagcca tccagtttac tttgcagggc 1020 ttcccaacct taccagaggtcgccccagct ggcaattccg gttcgcttgc tgtccataaa 1080 accgcccagt ctagctatcgccatgtaagc ccactgcaag ctacctgctt tctctttgcg 1140 cttgcgtttt cccttgtccagatagcccag tagtgacatt catccggggt cagcaccgtt 1200 tctgcggact ggctttctacgtgttccgct tcctttagca gcccttgcgc cctgagtgct 1260 tgcggcagcg tgaagctgcttcctcgctca ctgactcgct gcgctcggtc gttcggctgc 1320 ggcgagcggt atcagctcactcaaaggcgg taatacggtt atccacagaa tcaggggata 1380 acgcaggaaa gaacatgtgagcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 1440 cgttgctggc gtttttccataggctccgcc cccctgacga gcatcacaaa aatcgacgct 1500 caagtcagag gtggcgaaacccgacaggac tataaagata ccaggcgttt ccccctggaa 1560 gctccctcgt gcgctctcctgttccgaccc tgccgcttac cggatacctg tccgcctttc 1620 tcccttcggg aagcgtggcgctttctcaat gctcacgctg taggtatctc agttcggtgt 1680 aggtcgttcg ctccaagctgggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 1740 ccttatccgg taactatcgtcttgagtcca acccggtaag acacgactta tcgccactgg 1800 cagcagccac tggtaacaggattagcagag cgaggtatgt aggcggtgct acagagttct 1860 tgaagtggtg gcctaactacggctacacta gaaggacagt atttggtatc tgcgctctgc 1920 tgaagccagt taccttcggaaaaagagttg gtagctcttg atccggcaaa caaaccaccg 1980 ctggtagcgg tggtttttttgtttgcaagc agcagattac gcgcagaaaa aaaggatctc 2040 aagaagatcc tttgatcttttctacggggt ctgacgc 2077 8 33 DNA Artificial Sequence PCR primer foramplifying a 3′-UTR/poly(A) segment. 8 ccgagggcaa aggaataggc gggactctggggt 33 9 21 DNA Artificial Sequence PCR primer for amplifying a3′-UTR/poly(A) segment. 9 ctcgaggtcg acgggatcca g 21 10 33 DNAArtificial Sequence PCR primer for amplifying a hygromycin codingregion. 10 ggatgaggat cgtttcgcat gaaaaagcct gaa 33 11 33 DNA ArtificialSequence PCR primer for amplifying a hygromycin coding region. 11accccagagt cccgcctatt cctttgccct cgg 33 12 20 DNA Artificial SequencePCR primer for amplifying an amp/SV40 early promoter. 12 cgtcaggtggcacttttcgg 20 13 33 DNA Artificial Sequence PCR primer for amplifyingthe amp/SV40 early promoter. 13 ttcaggcttt ttcatgcgaa acgatcctca tcc 3314 8705 DNA Artificial Sequence pShuttle 14 tcgagcatga ccaaaatcccttaacgtgag ttttcgttcc actgagcgtc agaccccgta 60 gaaaagatca aaggatcttcttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 120 acaaaaaaac caccgctaccagcggtggtt tgtttgccgg atcaagagct accaactctt 180 tttccgaagg taactggcttcagcagagcg cagataccaa atactgtcct tctagtgtag 240 ccgtagttag gccaccacttcaagaactct gtagcaccgc ctacatacct cgctctgcta 300 atcctgttac cagtggctgctgccagtggc gataagtcgt gtcttaccgg gttggactca 360 agacgatagt taccggataaggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 420 cccagcttgg agcgaacgacctacaccgaa ctgagatacc tacagcgtga gcattgagaa 480 agcgccacgc ttcccgaagggagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 540 acaggagagc gcacgagggagcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 600 gggtttcgcc acctctgacttgagcgtcga tttttgtgat gctcgtcagg ggggcggagc 660 ctatggaaaa acgccagcaacgcggccttt ttacggttcc tggccttttg ctggcctttt 720 gctcacatgt tctttcctgcgttatcccct gattctgtgg ataaccgtat taccgccttt 780 gagtgagctg ataccgctcgccgcagccga acgaccgagc gcagcgagtc agtgagcgag 840 gaagccgtca ggtggcacttttcggggaaa tgtgcgcgga acccctattt gtttattttt 900 ctaaatacat tcaaatatgtatccgctcat gagacaataa ccctgataaa tgcttcaata 960 atattgaaaa aggaagagtcctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta 1020 gggtgtggaa agtccccaggctccccagca ggcagaagta tgcaaagcat gcatctcaat 1080 tagtcagcaa ccaggtgtggaaagtcccca ggctccccag caggcagaag tatgcaaagc 1140 atgcatctca attagtcagcaaccatagtc ccgcccctaa ctccgcccat cccgccccta 1200 actccgccca gttccgcccattctccgccc catggctgac taattttttt tatttatgca 1260 gaggccgagg ccgcctcggcctctgagcta ttccagaagt agtgaggagg cttttttgga 1320 ggcctaggct tttgcaaagatcgatcaaga gacaggatga ggatcgtttc gcatgaaaaa 1380 gcctgaactc accgcgacgtctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc 1440 cgacctgatg cagctctcggagggcgaaga atctcgtgct ttcagcttcg atgtaggagg 1500 gcgtggatat gtcctgcgggtaaatagctg cgccgatggt ttctacaaag atcgttatgt 1560 ttatcggcac tttgcatcggccgcgctccc gattccggaa gtgcttgaca ttggggaatt 1620 cagcgagagc ctgacctattgcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct 1680 gcctgaaacc gaactgcccgctgttctgca gccggtcgcg gaggccatgg atgcgatcgc 1740 tgcggccgat cttagccagacgagcgggtt cggcccattc ggaccgcaag gaatcggtca 1800 atacactaca tggcgtgatttcatatgcgc gattgctgat ccccatgtgt atcactggca 1860 aactgtgatg gacgacaccgtcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct 1920 ttgggccgag gactgccccgaagtccggca cctcgtgcac gcggatttcg gctccaacaa 1980 tgtcctgacg gacaatggccgcataacagc ggtcattgac tggagcgagg cgatgttcgg 2040 ggattcccaa tacgaggtcgccaacatctt cttctggagg ccgtggttgg cttgtatgga 2100 gcagcagacg cgctacttcgagcggaggca tccggagctt gcaggatcgc cgcggctccg 2160 ggcgtatatg ctccgcattggtcttgacca actctatcag agcttggttg acggcaattt 2220 cgatgatgca gcttgggcgcagggtcgatg cgacgcaatc gtccgatccg gagccgggac 2280 tgtcgggcgt acacaaatcgcccgcagaag cgcggccgtc tggaccgatg gctgtgtaga 2340 agtactcgcc gatagtggaaaccgacgccc cagcactcgt ccgagggcaa aggaataggc 2400 gggactctgg ggttcgaaatgaccgaccaa gcgacgccca acctgccatc acgagatttc 2460 gattccaccg ccgccttctatgaaaggttg ggcttcggaa tcgttttccg ggacgccggc 2520 tggatgatcc tccagcgcggggatctcatg ctggagttct tcgcccaccc caacttgttt 2580 attgcagctt ataatggttacaaataaagc aatagcatca caaatttcac aaataaagca 2640 tttttttcac tgcattctagttgtggtttg tccaaactca tcaatgtatc ttatcatgtc 2700 tggatccgat gtacgggccagatatacgcg ttgacattga ttattgacta gttattaata 2760 gtaatcaatt acggggtcattagttcatag cccatatatg gagttccgcg ttacataact 2820 tacggtaaat ggcccgcctggctgaccgcc caacgacccc cgcccattga cgtcaataat 2880 gacgtatgtt cccatagtaacgccaatagg gactttccat tgacgtcaat gggtggacta 2940 tttacggtaa actgcccacttggcagtaca tcaagtgtat catatgccaa gtacgccccc 3000 tattgacgtc aatgacggtaaatggcccgc ctggcattat gcccagtaca tgaccttatg 3060 ggactttcct acttggcagtacatctacgt attagtcatc gctattacca tggtgatgcg 3120 gttttggcag tacatcaatgggcgtggata gcggtttgac tcacggggat ttccaagtct 3180 ccaccccatt gacgtcaatgggagtttgtt ttggcaccaa aatcaacggg actttccaaa 3240 atgtcgtaac aactccgccccattgacgca aatgggcggt aggcgtgtac ggtgggaggt 3300 ctatataagc agagctctctggctaactag agaacccact gcttaactgg cttatcgaaa 3360 ttaatacgac tcactatagggagacccaag cttggtaccg agctcggatc cactagtaac 3420 ggccgccagt gtgctggaattctgcagata tccatcacac tggcggccgc tcgagcatgc 3480 atctagaggg ccctattctatagtgtcacc taaatgctag agctcgctga tcagcctcga 3540 ctgtgccttc tagttgccagccatctgttg tttgcccctc ccccgtgcct tccttgaccc 3600 tggaaggtgc cactcccactgtcctttcct aataaaatga ggaaattgca tcgcattgtc 3660 tgagtaggtg tcattctattctggggggtg gggtggggca ggacagcaag ggggaggatt 3720 gggaagacaa tagcaggcatgctggggatg cggtgggctc tatggcttct gaggcggaaa 3780 gaaccaggat cccccgccgccggacgaact aaacctgact acggcatctc tgccccttct 3840 tcgctggtac gaggagcgcttttgttttgt attggtcacg gggcagtgca tgtaatccct 3900 tcagttggtt ggtacaacttgccaactggg ccctgttcca catgtgacac ggggggggac 3960 caaacacaaa ggggttctctgactgtagtt gacatcctta taaatggatg tgcacatttg 4020 ccaacactga gtggctttcatcctggagca gactttgcag tctgtggact gcaacacaac 4080 attgccttta tgtgtaactcttggctgaag ctcttacacc aatgctgggg gacatgtacc 4140 tcccaggggc ccaggaagactacgggaggc tacaccaacg tcaatcagag gggcctgtgt 4200 agctaccgat aagcggaccctcaagagggc attagcaata gtgtttataa ggcccccttg 4260 ttaaccctaa acgggtagcatatgcttccc gggtagtagt atatactatc cagactaacc 4320 ctaattcaat agcatatgttacccaacggg aagcatatgc tatcgaatta gggttagtaa 4380 aagggtccta aggaacagcgatatctccca ccccatgagc tgtcacggtt ttatttacat 4440 ggggtcagga ttccacgagggtagtgaacc attttagtca caagggcagt ggctgaagat 4500 caaggagcgg gcagtgaactctcctgaatc ttcgcctgct tcttcattct ccttcgttta 4560 gctaatagaa taactgctgagttgtgaaca gtaaggtgta tgtgaggtgc tcgaaaacaa 4620 ggtttcaggt gacgcccccagaataaaatt tggacggggg gttcagtggt ggcattgtgc 4680 tatgacacca atataaccctcacaaacccc ttgggcaata aatactagtg taggaatgaa 4740 acattctgaa tatctttaacaatagaaatc catggggtgg ggacaagccg taaagactgg 4800 atgtccatct cacacgaatttatggctatg ggcaacacat aatcctagtg caatatgata 4860 ctggggttat taagatgtgtcccaggcagg gaccaagaca ggtgaaccat gttgttacac 4920 tctatttgta acaaggggaaagagagtgga cgccgacagc agcggactcc actggttgtc 4980 tctaacaccc ccgaaaattaaacggggctc cacgccaatg gggcccataa acaaagacaa 5040 gtggccactc ttttttttgaaattgtggag tgggggcacg cgtcagcccc cacacgccgc 5100 cctgcggttt tggactgtaaaataagggtg taataacttg gctgattgta accccgctaa 5160 ccactgcggt caaaccacttgcccacaaaa ccactaatgg caccccgggg aatacctgca 5220 taagtaggtg ggcgggccaagataggggcg cgattgctgc gatctggagg acaaattaca 5280 cacacttgcg cctgagcgccaagcacaggg ttgttggtcc tcatattcac gaggtcgctg 5340 agagcacggt gggctaatgttgccatgggt agcatatact acccaaatat ctggatagca 5400 tatgctatcc taatctatatctgggtagca taggctatcc taatctatat ctgggtagca 5460 tatgctatcc taatctatatctgggtagta tatgctatcc taatttatat ctgggtagca 5520 taggctatcc taatctatatctgggtagca tatgctatcc taatctatat ctgggtagta 5580 tatgctatcc taatctgtatccgggtagca tatgctatcc taatagagat tagggtagta 5640 tatgctatcc taatttatatctgggtagca tatactaccc aaatatctgg atagcatatg 5700 ctatcctaat ctatatctgggtagcatatg ctatcctaat ctatatctgg gtagcatagg 5760 ctatcctaat ctatatctgggtagcatatg ctatcctaat ctatatctgg gtagtatatg 5820 ctatcctaat ttatatctgggtagcatagg ctatcctaat ctatatctgg gtagcatatg 5880 ctatcctaat ctatatctgggtagtatatg ctatcctaat ctgtatccgg gtagcatatg 5940 ctatcctcat gcatatacagtcagcatatg atacccagta gtagagtggg agtgctatcc 6000 tttgcatatg ccgccacctcccaagggggc gtgaattttc gctgcttgtc cttttcctgc 6060 tggttgctcc cattcttaggtgaatttaag gaggccaggc taaagccgtc gcatgtctga 6120 ttgctcacca ggtaaatgtcgctaatgttt tccaacgcga gaaggtgttg agcgcggagc 6180 tgagtgacgt gacaacatgggtatgcccaa ttgccccatg ttgggaggac gaaaatggtg 6240 acaagacaga tggccagaaatacaccaaca gcacgcatga tgtctactgg ggatttattc 6300 tttagtgcgg gggaatacacggcttttaat acgattgagg gcgtctccta acaagttaca 6360 tcactcctgc ccttcctcaccctcatctcc atcacctcct tcatctccgt catctccgtc 6420 atcaccctcc gcggcagccccttccaccat aggtggaaac cagggaggca aatctactcc 6480 atcgtcaaag ctgcacacagtcaccctgat attgcaggta ggagcgggct ttgtcataac 6540 aaggtcctta atcgcatccttcaaaacctc agcaaatata tgagtttgta aaaagaccat 6600 gaaataacag acaatggactcccttagcgg gccaggttgt gggccgggtc caggggccat 6660 tccaaagggg agacgactcaatggtgtaag acgacattgt ggaatagcaa gggcagttcc 6720 tcgccttagg ttgtaaagggaggtcttact acctccatat acgaacacac cggcgaccca 6780 agttccttcg tcggtagtcctttctacgtg actcctagcc aggagggccc ttaaaccttc 6840 tgcaatgttc tcaaatttcgggttggaacc tccttgacca cgatgctttc caaaccaccc 6900 tccttttttg cgcctgcctccatcaccctg accccggggt ccagtgcttg ggccttctcc 6960 tgggtcatct gcggggccctgctctatcgc tcccgggggc acgtcaggct caccatctgg 7020 gccaccttct tggtggtattcaaaataatc ggcttcccct acagggtgga aaaatggcct 7080 tctacctgga gggggcctgcgcggtggaga cccggatgat gatgactgac tactgggact 7140 cctgggcctc ttttctccacgtccacgacc tctccccctg gctctttcac gacttccccc 7200 cctggctctt tcacgtcctctaccccggcg gcctccacta cctcctcgac cccggcctcc 7260 actacctcct cgaccccggcctccactgcc tcctcgaccc cggcctccac ctcctgctcc 7320 tgcccctcct gctcctgcccctcctcctgc tcctgcccct cctgcccctc ctgctcctgc 7380 ccctcctgcc cctcctgctcctgcccctcc tgcccctcct gctcctgccc ctcctgcccc 7440 tcctcctgct cctgcccctcctgcccctcc tcctgctcct gcccctcctg cccctcctgc 7500 tcctgcccct cctgcccctcctgctcctgc ccctcctgcc cctcctgctc ctgcccctcc 7560 tgctcctgcc cctcctgctcctgcccctcc tgctcctgcc cctcctgccc ctcctgcccc 7620 tcctcctgct cctgcccctcctgctcctgc ccctcctgcc cctcctgccc ctcctgctcc 7680 tgcccctcct cctgctcctgcccctcctgc ccctcctgcc cctcctcctg ctcctgcccc 7740 tcctgcccct cctcctgctcctgcccctcc tcctgctcct gcccctcctg cccctcctgc 7800 ccctcctcct gctcctgcccctcctgcccc tcctcctgct cctgcccctc ctcctgctcc 7860 tgcccctcct gcccctcctgcccctcctcc tgctcctgcc cctcctcctg ctcctgcccc 7920 tcctgcccct cctgcccctcctgcccctcc tcctgctcct gcccctcctc ctgctcctgc 7980 ccctcctgct cctgcccctcccgctcctgc tcctgctcct gttccaccgt gggtcccttt 8040 gcagccaatg caacttggacgtttttgggg tctccggaca ccatctctat gtcttggccc 8100 tgatcctgag ccgcccggggctcctggtct tccgcctcct cgtcctcgtc ctcttccccg 8160 tcctcgtcca tggttatcaccccctcttct ttgaggtcca ctgccgccgg agccttctgg 8220 tccagatgtg tctcccttctctcctaggcc atttccaggt cctgtacctg gcccctcgtc 8280 agacatgatt cacactaaaagagatcaata gacatcttta ttagacgacg ctcagtgaat 8340 acagggagtg cagactcctgccccctccaa cagccccccc accctcatcc ccttcatggt 8400 cgctgtcaga cagatccaggtctgaaaatt ccccatcctc cgaaccatcc tcgtcctcat 8460 caccaattac tcgcagcccggaaaactccc gctgaacatc ctcaagattt gcgtcctgag 8520 cctcaagcca ggcctcaaattcctcgtccc cctttttgct ggacggtagg gatggggatt 8580 ctcgggaccc ctcctcttcctcttcaaggt caccagacag agatgctact ggggcaacgg 8640 aagaaaagct gggtgcggcctgtgaggatc agcttatcga tgataagctg tcaaacatga 8700 gaatt 8705 15 29 DNAArtificial Sequence CatCass1 15 ctgatgaggt cgcgactagt gttgacaat 29 16 27DNA Artificial Sequence CatCass2 16 ttcggtctcg cgagcaggtt agtgaca 27 175658 DNA Artificial Sequence pBK 17 ctagttctgg cgcagaacca tggcctttgtccagtttaac tggggacaag gccaagattc 60 ctaggctcgc aaaacatgtc tgtcatgcactttccttcct gaggtcatgg tttggctgca 120 ttccatgggt aagcagctcc tccctgtgagtcatgcactt tccttcctga ggtcatggtt 180 tggctgcatt cccctgtgag tcatgcactttccttcctga ggtcatggtt tggctgcatt 240 ccatgggtaa gcagctcctc cctgtggcctttttttttat aatatataag aggccgaggc 300 cgcctctgcc tccacccttt ctctcaagtagtaagggtgt ggaggctttt tctgaggcct 360 agcaaaacta tttggggaaa tccctattcttttgcaattt ttgcaaaaat ggataaagtt 420 cttaacaggg aagaatccat ggagctcatggaccttttag gccttgaaag agctgcctgg 480 ggaaatcttc ccttaatgag aaaagcttatttaaggaagt gtaaggaatt tcatcctgac 540 aaagggggcg acgaggataa aatgaagagaatgaatactt tgtataaaaa aatggagcag 600 gatgtaaagg tagctcatca gcctgattttggaacttgga gtagctcaga ggtttgtgct 660 gattttcctc tttgcccaga taccctgtactgcaaggaat ggcctatttg ttccaaaaag 720 ccttctgtgc actgcccttg catgctatgtcagcttagat taaggcattt aaatagaaaa 780 tttttaagaa aagagccctt ggtttggatagattgctact gcattgactg cttcacacag 840 tggtttggct tagacctaac tgaagaaactctgcaatggt gggtccaaat aattggagaa 900 actcccttca gagatctaaa gctttaaggtaactaactta tatttagata aataataaaa 960 tattaaaagg ccctaagtaa ttattttttttataggtgcc aacctatgga acagaagagt 1020 gggagtcctg gtggagttcc tttaatgaaaaatgggatga agatttattt tgccatgaag 1080 atatgtttgc cagtgatgaa gaagcaacagcagattctca acactcaaca ccacccaaaa 1140 aaaaaagaaa ggtagaagac cctaaagactttccctctga tctacaccag tttcttagtc 1200 aagctgtatt tagtaataga acccttgcctgctttgctgt gtatactact aaagaaaaag 1260 ctcaaattct gtataaaaaa cttatggaaaaatattctgt aacttttatt agtagacaca 1320 tgtgtgctgg gcataatatt atattctttttaactccaca tagacataga gtttctgcaa 1380 ttaataattt ctgtcaaaag ctgtgtacctttagtttttt aatttgtaag ggtgttaata 1440 aggaatactt actatatagt gccttaactagagatccata ccatactata gaagaaagca 1500 ttcaaggggg cttaaaggag catgattttagcccagaaga gcctgaagaa acaaagcagg 1560 tgtcttggaa attaattact gagtatgcagtagagacaaa gtgtgaggat gtgtttttat 1620 tattaggtat gtatttagaa tttcaatacaatgtagagga gtgtaaaaag tgtcagaaaa 1680 aagaccagcc ttatcacttt aagtatcatgaaaagcactt tgcaaatgct attatttttg 1740 cagaaagtaa aaatcaaaaa agtatttgtcagcaagcagt agatacagtt ttagctaaaa 1800 aaagagtaga tacccttcat atgaccagggaagaaatgct aacagaaaga ttcaatcata 1860 tattagataa aatggattta atatttggagctcatggaaa tgctgtacta gaacaatata 1920 tggcaggtgt tgcttggctg cactgtttgctacctaaaat ggattctgta atatttgatt 1980 ttttgcactg tattgttttc aatgtacctaaaagaagata ctggttattt aaaggtccca 2040 ttgatagtgg aaaaacaaca ctagctgccgggttattaga tttgtgtggt ggtaaagcct 2100 taaatgtaaa cctacccatg gaaaggctaacctttgagct aggtgtagct atagatcagt 2160 acatggttgt ttttgaagat gtaaaagggacaggagctga atcaaaggat ttgccttcag 2220 gacatggaat aaacaattta gacagtttgagagattattt agatggaagt gttaaggtaa 2280 atttagaaaa gaaacattta aacaaaagaacccaaatatt tccaccaggc ttggttacaa 2340 tgaatgagta tcctgtccct aaaaccctgcaagctagatt tgtaagacaa atagatttta 2400 ggcccaaaat atatttaaga aaatccttacaaaactcaga gttcttactt gaaaaaagaa 2460 ttttacaaag tggaatgacc ttgttgctactgctaatttg gtttaggcct gtagctgatt 2520 ttgcaactga tatacaatct agaattgttgaatggaagga aaggctggat tctgagataa 2580 gtatgtatac tttttcaagg atgaaatataatatatgctt ggggaaatgt attcttgata 2640 ttacaagaga agaggattca gaaactgaagactctggaca tggatcaagc actgaatccc 2700 aatcacaatg ctcttcccaa gtctcagatacttcagcccc tgctgaagat tcccaaaggt 2760 cagaccccca tagtcaagag ttgcatttgtgtaaaggctt tcagtgtttt aaaaggccta 2820 aaacaccacc cccaaaataa cacaagcttaaaagtggctt atacaaaagc agcatttatt 2880 aaatgtatat gtacaataaa agcacctgtttaaagcattt tggtttgcaa ttgtccctgt 2940 ttgtcaatat atcttatcat atctgggtcccctggaagta actagatgat ccgctgtgga 3000 atgtgtgtca gttagggtgt ggaaagtccccaggctcccc agcaggcaga agtatgcaaa 3060 gcatctcaat tagtcagcaa ccaggtgtggaaagtcccca ggctccccag caggcagaag 3120 tatgcaaagt aatagtaatc aattacggggtcattagttc atagcccata tatggagttc 3180 cgcgttacat aacttacggt aaatggcccgcctggctgac cgcccaacga cccccgccca 3240 ttgacgtcaa taatgacgta tgttcccatagtaacgccaa tagggacttt ccattgacgt 3300 caatgggtgg agtatttacg gtaaactgcccacttggcag tacatcaagt gtatcatatg 3360 ccaagtacgc cccctattga cgtcaatgacggtaaatggc ccgcctggca ttatgcccag 3420 tacatgacct tatgggactt tcctacttggcagtacatct acgtattagt catcgctatt 3480 accatggcga tgcggttttg gcagtacatcaatgggcgtg gatagcggtt tgactcacgg 3540 ggatttccaa gtctccaccc cattgacgtcaatgggagtt tgttttggca ccaaaatcaa 3600 cgggactttc caaaatgtcg taacaactccgccccattga cgcaaatggg cggtaggcgt 3660 gtacggtggg aggtctatat aagcagagctggtttagtga accgtcagat ccgctagcgc 3720 taccggactc agatctcgag ctcaagctaatcatcggcat agtatatcgg catagtataa 3780 tacgactcac tataggaggg ccaccatggccaagttgacc agtgccgttc cggtgcttac 3840 cgcgcgcgac gtcgccggag cggtcgagttctggaccgac cggctcgggt tctcccggga 3900 cttcgtggag gacgacttcg ccggtgtggtccgggacgac gtgaccctgt tcatcagcgc 3960 ggtccaggac caggtggtgc cggacaacaccctggcctgg gtgtgggtgc gcggcctgga 4020 cgagctgtac gccgagtggt cggaggtcgtgtccacgaac ttccgggacg cctccgggcc 4080 ggccatgacc gagatcggcg agcagccgtgggggcgggag ttcgccctgc gcgacccggc 4140 cggcaactgc gtgcacttcg tggccgaggagcaggactga ccgacgccga ccaacaccgc 4200 cggggggagg ctaactgaaa cacggaaggagacaataccg gaaggaaccc gcgctatgac 4260 ggcaataaaa agacagaata aaacgcacggtgttgggtcg tttgttcata aacgcggggt 4320 tcggtcccag ggctggcact ctgtcgataccccaccgacg gcggcccacg ggtcgaattg 4380 cgcttccctg atgagaccga aaggtcgaaagtcgaaagac tcggaagcga aagcttggtg 4440 atgcattcga tatcgtttaa acgcccgggcgcggccgcgg cgcctccagt cgacgaaagt 4500 cggtctgccg aaaggcactg atgagtccgaaaggacgaaa ccgacttgct agataactga 4560 tcataatcag ccataccaca tttgtagaggttttacttgc tttaaaaaac ctcccacacc 4620 tccccctgaa cctgaaacat aaaatgaatgcaattgttgt tgttaacttg tttattgcag 4680 cttataatgg ttacaaataa agcaatagcatcacaaattt cacaaataaa gcattttttt 4740 cactgcattc tagttgtggt ttgtccaaactcatcaatgt atcttaacgc gtaaattgta 4800 agcgttaatc atgcggccca tgaccaaaatcccttaacgt gagttttcgt tccactgagc 4860 gtcagacccc gtagaaaaga tcaaaggatcttcttgagat cctttttttc tgcgcgtaat 4920 ctgctgcttg caaacaaaaa aaccaccgctaccagcggtg gtttgtttgc cggatcaaga 4980 gctaccaact ctttttccga aggtaactggcttcagcaga gcgcagatac caaatactgt 5040 ccttctagtg tagccgtagt taggccaccacttcaagaac tctgtagcac cgcctacata 5100 cctcgctctg ctaatcctgt taccagtggctgctgccagt ggcgataagt cgtgtcttac 5160 cgggttggac tcaagacgat agttaccggataaggcgcag cggtcgggct gaacgggggg 5220 ttcgtgcaca cagcccagct tggagcgaacgacctacacc gaactgagat acctacagcg 5280 tgagcattga gaaagcgcca cgcttcccgaagggagaaag gcggacaggt atccggtaag 5340 cggcagggtc ggaacaggag agcgcacgagggagcttcca gggggaaacg cctggtatct 5400 ttatagtcct gtcgggtttc gccacctctgacttgagcgt cgatttttgt gatgctcgtc 5460 aggggggcgg agcctatgga aaaacgccagcaacgcggcc tttttacggt tcctggcctt 5520 ttgctggcct tttgctcaca tgttctttcctgcgttatcc cctgattctg tggataaccg 5580 tattaccgcc tttgagtgag ctgataccgctcgccgcagc cgaacgaccg agcgcagcga 5640 gtcagtgagc gaggaagc 5658 18 178DNA Artificial Sequence Multi-cloning sequence flanked by two cis-actingribozymes (CAR′s). 18 gagctcgctt ccctgatgag tccgaaagga cgaaagtcgaaagactcgga agcgaaagct 60 tggtgatgca ttcgatatcg tttaaacgcc cgggcgcggccgcggcgcct ccagtcgacg 120 aaagtcggtc tgccgaaagg cactgatgag tccgaaaggacgaaaccgac ttggtacc 178 19 14 DNA herpes simplex virus 19 cgacgccgcccgcc 14 20 13 DNA herpes simplex virus 20 ctgcgcgcgt ggc 13 21 14 DNAherpes simplex virus 21 gcgcctgcgc gggg 14 22 14 DNA herpes simplexvirus 22 cgccgccgac gcgc 14 23 13 DNA herpes simplex virus 23 ccccctccccgcg 13 24 14 DNA herpes simplex virus 24 gtggccgtgt cgcg 14 25 14 DNAherpes simplex virus 25 gccacacggc ggcg 14 26 14 DNA herpes simplexvirus 26 cgccgcgcgg tgcg 14 27 14 DNA herpes simplex virus 27 cgccgcgcggtgcg 14 28 15 DNA herpes simplex virus 28 ccccctgcgc gcctc 15 29 15 DNAherpes simplex virus 29 ggtggtgctg tactc 15 30 14 DNA herpes simplexvirus 30 gggcccgcgg tgtc 14 31 14 DNA herpes simplex virus 31 cctggcgtgcgagc 14 32 21 DNA herpes simplex virus 32 ggggaccacc gacgccatgg c 21 3314 DNA herpes simplex virus 33 cgtggcgctg gggc 14 34 14 DNA herpessimplex virus 34 cgggattcgc tggg 14 35 15 DNA Artificial SequencePortion of a multiple cloning site for use in making deletion libraries.35 ggtgatgcat tcgat 15 36 38 DNA Artificial Sequence Portion of amultiple cloning site for use in making deletion libraries. 36atcgtttaaa cgcccgggcg cggccgcggc gcctccag 38 37 10 DNA ArtificialSequence Portion of a multiple cloning site for use in making deletionlibraries. 37 ctggaggcgc 10 38 22 DNA Artificial Sequence Portion of anintermediate in the making of a deletion library, including a portion ofa multiple cloning site. 38 nnnnnnnnnn nnnnnnctcc ag 22 39 25 DNAArtificial Sequence 14 bp variable sequence fragment of a deletionlibrary including flanking portions of multiple cloning site. 39ggtgannnnn nnnnnnnnnc tccag 25 40 35 RNA Artificial Sequence Ahammerhead ribozyme comprising a catalytic core flanked by variablerecognition domains. 40 nnnnnnncug augaggucgc gagaccgaaa nnnnn 35 41 14RNA Artificial Sequence A target substrate comprising variable sequenceregions flanking a cleavage site. 41 nnnnnuhnnn nnnn 14 42 12 DNAArtificial Sequence A portion of an antisense library including an HphIsite. 42 ggtgannnnn nn 12 43 12 DNA Artificial Sequence A portion of anantisense library including a BpmI site. 43 nnnnnnctcc ag 12 44 15 DNAArtificial Sequence A sequence flanking a chloramphenicol (CAT) gene andcontaining an NruI site. 44 ctgatgaggt cgcga 15 45 14 DNA ArtificialSequence A sequence flanking a chloramphenicol (CAT) gene and containingan NruI site. 45 tcgcgagagc cgaa 14 46 27 DNA Artificial SequenceSequence flanking a chloramphenicol (CAT) gene after insertion of theantisense library. 46 ggtgannnnn nnctgatgag gtcgcga 27 47 25 DNAArtificial Sequence Sequence flanking the chloramphenicol (CAT) geneafter insertion into the antisense library. 47 tcgcgagacc gaannnnnnctccag 25 48 46 DNA Artificial Sequence Hammerhead ribozyme library withflanking sequences. 48 ggtgannnnn nnctgatgag gtcgcgagac cgaannnnnnctccag 46 49 20 DNA Artificial Sequence Deletion fragment in a deletionfragment library, including a portion of a multiple cloning site. 49nnnnnnnnnn nnnnctccag 20 50 53 DNA Artificial Sequence Multiple cloningsite for use in making deletion libraries. 50 tgcattcgat atcgtttaaacgcccgggcg cggccgcggc gcctccagtc gac 53

The subject matter claimed is:
 1. A method for identifying target sitesfor antisense-mediated inhibition of a selected gene comprising: (a)constructing a directed antisense library targeted at said selected genewherein said library is contained in a cloning vector having a promoterconfigured for transcribing antisense transcripts from said directedantisense library in vitro; (b) transcribing antisense transcripts fromsaid directed antisense library in vitro; (c) incubating said antisensetranscripts with a lysate from a cell containing target transcriptstranscribed from said selected gene such that antisense transcriptstargeted to the target transcripts bind to such target transcripts; and(d) analyzing the antisense transcripts that bind the target transcriptand determining a target site on the antisense transcript that isassociated with binding of the target transcript.
 2. The method of claim1 wherein constructing a directed antisense library targeted at theselected gene comprises: (a) preparing a double-stranded cDNA,comprising a first end, a second end, and a central site thereof, fromthe target transcript and cloning the cDNA in a cloning vectorcomprising a promoter configured such that an antisense transcript ofthe cDNA is synthesized upon transcription mediated by the promoter,resulting in a cloned cDNA; (b) creating a plurality of deletionderivatives of the cloned cDNA wherein each of the plurality of deletionderivatives has a deletion extending from the first end into the clonedcDNA such that the plurality of deletion derivatives comprises adeletion library comprising deletions extending serially into the cDNA;(c) reducing the size of the cDNA contained in the deletion library to apreselected size by removing a portion of the cDNA from the second endthereof to result in a fragment library; (d) inserting an antisense geneDNA into the central site of the cDNA in the fragment library, therebyobtaining the antisense library.
 3. The method of claim 2 wherein saidplurality of deletion derivatives is created with exonuclease IIIresection of the cloned cDNA.
 4. The method of claim 2 wherein thereducing the size of the cDNA contained in the deletion library to apreselected size comprises digesting the deletion library with a typeIIS restriction endonuclease.
 5. The method of claim 2 wherein theinserting an antisense gene DNA into the central site of the cDNA in thefragment library comprises digesting the fragment library with a typeIIS restriction endonuclease, thereby creating the central site, andligating the antisense gene DNA at the central site.
 6. The method ofclaim 2 wherein the antisense gene DNA comprises a ribozyme catalyticcore.
 7. The method of claim 2 wherein the ribozyme catalytic core is ahammerhead ribozyme catalytic core.
 8. The method of claim 1 wherein thecloning vector is pShuttle (SEQ ID NO:14).
 9. The method of claim 1wherein the cloning vector is pBK (SEQ ID NO: 17).